The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
Nucleic acid detection in a biological sample such as whole blood often requires that the nucleic acid is extracted and purified from the sample prior to performing PCR. This is because constituents, such as hemoglobin in blood samples, and preserving reagents, such as anticoagulants, can interfere with PCR amplification (Wang, J-T., et al., 1992, J. Clin. Microbiol. 30:750). Target nucleic acids are particularly difficult to detect because the nucleic acids are typically present at much lower levels than endogenous nucleic acids such as genomic DNA or RNA transcribed therefrom. Extraction of nucleic acids of organisms from biological samples is time consuming and involves a high risk of contamination.
Given the high degree of complexity associated with isolating and detecting a nucleic acid molecule in a biological sample, it becomes increasingly desirable to detect the nucleic acid directly in a biological sample without any upstream nucleic acid extraction or extensive pre-processing step. Several methods have been reported for direct PCR of a pathogenic nucleic acid from blood samples, such as microwave irradiation (Ihhara, M., et al., 1994, BioTechniques 17(4):726), hydrogen peroxide treatment (Rudbeck, L. and Dissing, J., 1998, BioTechniques 25(4):588), and sodium hydroxide treatment (Queipo-Ortuna, M., et al., 1999, BioTechniques 27(2): 248). However, in cases where a quick diagnosis is sought, there is a need for quick methods that involve only a few steps and minimal technological requirements, and that still achieve consistent successful amplification of a nucleic acid in a biological sample.